Bmp 2

To determine the osteogenic stimulatory effect of BMP-2 and FGF-2 on BM-MSCs, the cells were treated with 100 ng/mL of BMP-2 (Stem Cell Technologies, Grenoble, France, cat. no. 78004.1) and/or 20 ng/mL FGF-2 (Merck, Saint Louis, MO, USA, cat. no. F0291). BM-MSCs cultured in the αMEM complete medium were used as a control. The supplemented and control media were changed every three days. The experiments on osteogenic induced BM-MSCs were performed after 7, 14, and 21 days of incubation.

ncl^+/+ and ncl^−/− embryos were treated with 0.0625, 0.25 and 1 ng/μl human recombinant FGF8 (Thermo Fisher Scientific, PHG0184) as well as 1 ng/μl FGF8 along with 2 ng/μl BMH21 (Sigma-Aldrich, SML1183) and 1 μg/μl BMP2 (Stemcell Technologies, 78004) diluted in E2 media by immersion starting at 18 hpf. In addition, ncl^+/+ and ncl^−/− embryos were treated with 1 ng/μl FGF8 by immersion beginning at 30 hpf. The drugs in the media were replaced every 24 h until 5 dpf. The FGF8-treated larvae were added to the tank system and allowed to develop until 15 dpf.

The process of BM-MSC preparation was carried out at the Laboratory of Biology and Neoplastic Cells, Hirszweld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. A detailed description of the isolation and stimulation of BM-MSCs was described in a study by Gromolak et al. [24 (link)]. Briefly, four weeks before implantation of scaffolds, a portion of the bone marrow was aspirated from the iliac crest of the analgised experimental sheep. Within 30 min of bone marrow collection, BM-MSCs were harvested by centrifugation using a Lymphoflot (Bio-Rad, Dreieich, Germany) density gradient. The cells were washed with PBS and suspended in minimum essential medium α-transformation (αMEM, HIIET PAS, Wroclaw, Poland) supplemented with fetal bovine serum (Biowest, Riverside, MO, USA), L-glutamine and antibiotics, as well as 20 ng/mL of fibroblast growth factor-2 (FGF-2, Merck, Kenilworth, NJ, USA) and 100 ng/mL of bone morphogenetic protein-2 (BMP-2, Stem Cell Technologies, Saint-Egrève, France). Finally, 2 ml of autologous serum containing 20 million stimulated BM-MSCs was prepared for each experimental sheep.